Abstract

Sexual antagonism, or conflict between the sexes, has been proposed as a driving force in both sex chromosome turnover and speciation. Although closely related species often have different sex chromosome systems, it is unknown whether sex chromosome turnover contributes to the evolution of reproductive isolation between species. In this study, we show that a newly evolved sex chromosome harbours genes that contribute to speciation in threespine stickleback fish (Gasterosteus aculeatus). We first identified a neo-sex chromosome system found only in one member of a sympatric species pair in Japan. We then performed genetic linkage mapping of male-specific traits important for reproductive isolation between the Japanese species pair. The neo-X chromosome harbours loci for male courtship display traits that contribute to behavioural isolation, while the ancestral X chromosome contains loci for both behavioural isolation and hybrid male sterility. Our work not only provides strong evidence for a large-X effect on reproductive isolation in a vertebrate system, but also provides direct evidence that a young neo-X chromosome contributes to reproductive isolation between closely related species. Our data suggest that sex chromosome turnover might play a greater role in speciation than previously appreciated.

Sexually antagonistic selection has been proposed as a major driving force in the evolution of sex chromosomes. Specifically, natural selection is expected to favour linkage between genes with sexually antagonistic effects (i.e. beneficial in one sex and detrimental in the other sex) and the sex-determination locus, resulting in a reduction of recombination between sex chromosomes1. In an XY sex chromosome system, a reduction in recombination ultimately leads to the degeneration of the Y chromosome, thereby exposing alleles on the hemizygous X chromosome to selection in males2. Thus, male-beneficial alleles, manifested as sexually dimorphic and/or sexually selected traits, are predicted to accumulate on the X chromosome3,4. When these male-beneficial traits or alleles are important for reproductive isolation between species, the X-chromosome is also predicted to play an important role in speciation. A disproportionately large effect of the X-chromosome has been demonstrated for hybrid male sterility5,6, although the data supporting a large-X effect for other isolating barriers has been less consistent6.

Sexually antagonistic selection is also predicted to drive the divergence of sex chromosome systems between closely related species7,8. Sex chromosome turnover has been observed across many taxa, but is particularly striking in fishes9,10. Many independent groups of fishes show evidence for rapid evolution of sex chromosomes through several different mechanisms, including the transposition of an existing male-determination locus to an autosome11, the evolution of a new male-determination locus on an autosome12, and fusions between an autosome and an existing Y-chromosome13,14. It has been suggested that rapid turnover of sex chromosomes driven by sexual conflict might also play a role in the high speciation rates seen in some groups of fishes15–17. However, a direct role for sex chromosome turnover in speciation has not been empirically investigated in any system.

Here we demonstrate that there is a newly formed sex chromosome system in a threespine stickleback population found in Japan. This Japan Sea form diverged from the Pacific Ocean threespine stickleback during periods of geographical isolation between the Sea of Japan and the Pacific Ocean about 1.5–2 million years ago18,19. Because we can cross the derived Japan Sea form to the ancestral Pacific Ocean form, we have been able to take advantage of the genetic tools available for the threespine stickleback20 to investigate whether the evolution of a neo-sex chromosome has contributed to the evolution of male traits that act as isolating barriers between the derived Japan Sea and the ancestral Pacific Ocean populations.

Japan Sea stickleback neo-sex chromosome

In all threespine stickleback populations examined previously, including the Pacific Ocean form, linkage group (LG) 19 is the sex chromosome and LG9 is an autosome14,21,22. However, in the course of making a linkage map from a Japan Sea cross, we noticed that several LG9 markers co-segregated with LG19 markers previously found to be tightly linked to the sex-determination locus in a region of reduced recombination and rearrangements on the Y chromosome21,22. This association was observed when male meioses, but not female meioses, were analyzed (Fig. 1a). These data suggested that one copy of LG9 might be fused to one copy of LG19 (the Pacific Ocean Y chromosome), forming a neo-Y chromosome in Japan Sea sticklebacks. Therefore, we performed fluorescence in situ hybridization (FISH) with LG19 and LG9 probes on metaphase chromosome spreads from two different populations of the Japan Sea form. We found that Japan Sea males (n = 4) from two different populations have an odd number of chromosomes (2n = 41), with one large unpaired chromosome that hybridized to the LG19 and LG9 probes, providing evidence for the LG9-Y fusion (Fig. 1b; Supplementary Fig. 1). By contrast, Japan Sea females (n = 4) from both populations have an even number of chromosomes (2n = 42), and the LG9 and LG19 probes hybridize to separate chromosome pairs (Fig. 1b; Supplementary Fig. 1). Because the fused copy of LG9 segregates with the Y chromosome in Japan Sea males, the other copy segregates as an X chromosome; this neo-sex chromosome system is defined as an X1X2Y system13, in which X1 is the ancestral X chromosome (LG19) and X2 is the neo-X chromosome (LG9). Thus, the Japan Sea form has a unique neo-sex chromosome system, which has likely evolved within the past 1.5–2 million years of isolation between the Pacific Ocean and Japan Sea sticklebacks18,19.

Genetic and cytogenetic evidence of a fusion between one copy of LG9 and the Y-chromosome in Japan Sea males

Components of reproductive isolation

We have the opportunity to test the role of this neo-sex chromosome in reproductive isolation between the Japan Sea and the Pacific Ocean forms because both are anadromous and migrate into Lake Akkeshi and the Bekanbeushi marsh during the breeding season. In this location, they co-occur, along with hybrid adults and juveniles, within a hybrid zone in the Bekanbeushi River (Mid2 in Fig. 2; Supplementary Fig. 2). We found that temporal isolation, behavioural isolation, and hybrid male sterility contribute to reproductive isolation between the forms in the hybrid zone (Supplementary Fig. 3; Supplementary Discussion). We previously demonstrated that behavioural isolation and hybrid male sterility are asymmetric, but act in complementary directions19. Pacific Ocean females mate exclusively with Pacific Ocean males, while the Japan Sea females mate with both types of males in laboratory mate choice trials19. Although Japan Sea females do mate with Pacific Ocean males, hybrid males resulting from this cross have severely reduced fertility, while reciprocal hybrid males and all hybrid females are fertile19.

We found that a difference in male dorsal pricking behaviour also contributes to asymmetric behavioural isolation. Dorsal pricking is a component of male mating behaviour specific to threespine sticklebacks in which the male raises his dorsal spines and pricks the female. This behaviour might help the male assess the female, provide tactile stimulation to induce female spawning, or serve as a way for the male to display his dorsal spines to the female23,24. In Japan Sea males, the dorsal pricking display is greatly exaggerated, and the male pushes the female upwards during dorsal pricking (Fig. 3b)19. In addition, dorsal spine length is sexually dimorphic (males have longer spines than females) in the Japan Sea form25. By contrast, in the Pacific Ocean form, the dorsal pricking display is weak (Fig. 3b), and dorsal spine length is not sexually dimorphic19,25. We found that the Pacific Ocean females frequently escaped and did not resume mating after they encountered the aggressive dorsal pricking of Japan Sea males, while the Japan Sea females did not escape from males after dorsal pricking (Fig. 3b).

Our data demonstrate that loci important for both prezygotic and postzygotic isolation map to the X-chromosomes in this natural vertebrate system. This large X-effect is unlikely to result from an over-representation of these chromosomes in the stickleback genome, as LG9 and LG19 comprise just 9.0% of the stickleback genome (20.2 megabases (Mb)/446.6 Mb = 4.5% for each LG in the stickleback genome assembly; BROAD S1, Feb 2006). Because mapping in a backcross is likely to overestimate the effects of the hemizygous X26, we also performed QTL mapping of the same traits in an independent F2 intercross to ensure that the observed large X-effect was not simply the result of our backcross mapping strategy. We still detect QTL for dorsal pricking and first dorsal spine length on LG9 and QTL for male sterility and body size on LG19, with the addition of a single QTL for testis size on LG1 (Supplementary Fig. 6; Supplementary Table 2).

Discussion

Mapping of hybrid male sterility to the ancestral X-chromosome (LG19) is consistent with previous studies on hybrid male sterility in other systems5,6, suggesting that the large X-effect on hybrid male sterility is common across diverse taxa. Although the reasons for the large X-effect on hybrid sterility are debated5, in some cases, it results from genetic conflict in the form of sex ratio meiotic drive27,28. However, we find no evidence for sex ratio distortion in the weakly fertile F1 hybrid males resulting from a cross between Japan Sea females and Pacific Ocean males (Supplementary Table 3). We do, however, find evidence for conspecific epistasis between X-linked loci, which is commonly observed for hybrid male sterility in Drosophila29,30. Despite this finding of a common large X-effect across widely separated taxa, we found no evidence for any effect of the neo-X on hybrid male sterility. It may be that the relative age and/or levels of degeneration of sex chromosomes are an important factor in determining whether the X chromosome contributes to the evolution of hybrid male sterility.

Unlike male sterility, male courtship display traits conferring behavioural isolation between the Japan Sea and Pacific Ocean forms map to both the ancestral X chromosome and the neo-X chromosome. Interestingly, male body size is sexually dimorphic in both the Japan Sea and Pacific Ocean forms25 and maps to the ancestral X. By contrast, first dorsal spine length is only sexually dimorphic in the Japan Sea form25, and the dorsal pricking display is exaggerated in the Japan Sea males; these traits both map to the neo-X. Thus, these display traits might have evolved as a result of differential fitness effects between males and females specifically in the Japan Sea lineage. Although our cross design did not allow us to directly test whether these traits mapped to the neo-Y as predicted by theory7,8, it is possible that selection for linkage between male beneficial traits, such as dorsal pricking and dorsal spine length, and the sex-determination locus actually promoted the spread of the fusion between LG9 and the Y chromosome in the Japan Sea population7. Alternatively, these male beneficial traits may have accumulated on the neo-X chromosome3 after the formation of the neo-Y chromosome in the last 1.5–2 million years. In either case, our data provide direct empirical evidence linking sex chromosome turnover and reproductive isolation between closely related species.

Turnover of sex chromosomes between species is common in many groups of animals, where closely related species often differ in sex-determination and sex-chromosome systems9,10. Although it has been suggested that sex chromosome turnover might drive rapid speciation in cichlid fishes where a female colour trait is linked to an invading ZW sex chromosome system15–17, this hypothesis has not been directly tested. Given the potential role of sexual antagonism in driving both sex chromosome evolution1,3,7,8 and speciation31,32, we suggest that sex chromosome divergence between closely related species should be given further consideration as an important mechanism contributing to the evolution of reproductive isolation.

Methods Summary

Threespine sticklebacks of the Pacific Ocean and the Japan Sea form were collected with seine nets and minnow traps in Lake Akkeshi and the Beukanbeushi River in May-July of 2003–2008. Japan Sea fish were also collected from an allopatric site (Cape of Bankei) on the west coast of Hokkaido in 2008. For cytogenetic and behavioural studies, live fish were transported to the Fred Hutchinson Cancer Research Center; all experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC #1575). Cytogenetics and FISH were performed on Japan Sea males (n = 2) and a female (n = 1) from Akkeshi, as well as Japan Sea males (n = 2) and females (n = 3) from the allopatric site, using fluorescently labelled bacterial artificial chromosome (BAC) clones as previously described22. For population genetic analysis, 969 fish were genotyped with twelve neutral microsatellite markers (Supplementary Table 4). Mate choice experiments were conducted as previously described19. For QTL mapping, a Japan Sea female and a Pacific Ocean male were crossed to obtain an F1 hybrid family (J1 × P1). These F1 females were crossed with males resulting from a cross between a Pacific Ocean female and another Pacific Ocean male (P2 × P3) to generate backcross progeny. At maturity, 76 backcross males were phenotyped for traits related to body size, fertility, and dorsal pricking behaviour. These males were genotyped with LG9 and LG19 microsatellites, as well as a panel of SNP markers distributed across the stickleback genome (Supplementary Table 5). All DNA isolation and microsatellite genotyping were conducted previously described19; SNP genotyping was performed using Illumina Golden Gate arrays. The genotypes of 90 SNPs and 14 microsatellites were used to create a linkage map in JoinMap 3.033, and QTL analyses were performed in MapQTL 4.034 and R/qtl35.

Supplementary Material

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Acknowledgments

We thank M. Nishitani, J. Kitajima, M. Nishida, S. Takeyama, T. Andoh, T. Kuwahara, C. Torii, Akkeshi Fisheries Cooperative Association, Akkeshi Waterfowl Center, S. Brady, A. Southwick, all members of the Peichel lab, and many field assistants for technical help and discussion. We thank J. Boughman, T. Bradshaw, H. Malik, J. McKinnon, N. Phadnis, and D. Schluter for comments on the manuscript. We also thank the Broad Institute for the public release of an initial stickleback genome assembly. This research was supported by the Uehara Memorial Foundation (J.K.), a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Water and People Project of Research Institute for Humanity and Nature (S.M.), Akkeshi Town Grants-in-Aid for Scientific Research in the Lake Akkeshi-Bekanbeushi Wetland (M.K.), a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (C.L.P.), and National Institutes of Health grants T32 GM07270 (J.A.R.), R01 GM071854 (C.L.P.) and P50 HG02568 (R.M.M., D.M.K., and C.L.P.).

Footnotes

Supplementary Information including detailed methods, a supplementary discussion, seven figures, five tables, and associated references accompanies the paper on www.nature.com/nature.

Author Contributions J.K., S.M., and C.L.P. conceived and designed the study. F.C.J., Y.F.C., D.M.A., J.G., J.S., R.M.M., and D.M.K. contributed new reagents and carried out the SNP genotyping experiments for genome-wide linkage mapping. J.K, J.A.R., S.M., M.K., and C.L.P. performed all other experiments and analyzed the data. J.K. and C.L.P. wrote the manuscript.